Cross-linked glycopeptide-cephalosporin antibiotics

ABSTRACT

This invention provides cross-linked glycopeptides—cephalosporin compounds and pharmaceutically acceptable salts thereof which are useful as antibiotics. This invention also provides pharmaceutical compositions containing such compounds; methods for treating bacterial infections in a mammal using such compounds; and processes and intermediates useful for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/172,303, filed on Jun. 30, 2005 now U.S. Pat. No. 7,341,993; whichapplication is a continuation of U.S. application Ser. No. 10/269,471,filed Oct. 11, 2002 now U.S. Pat. No. 6,974,797; which applicationclaims the benefit of U.S. Provisional Application No. 60/328,889, filedon Oct. 12, 2001; the entire disclosures of which are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to novel cross-linkedvancomycin-cephalosporin compounds which are useful as antibiotics. Thisinvention is also directed to pharmaceutical compositions comprisingsuch compounds; methods of using such compounds as antibacterial agents;and processes and intermediates for preparing such compounds.

2. State of the Art

Various classes of antibiotic compounds are known in the art including,for example, β-lactam antibiotics, such as cephalosporins, andglycopeptide antibiotics, such as vancomycin. Cross-linked antibioticcompounds are also known in the art. See, for example, U.S. Pat. No.5,693,791, issued to W. L. Truett and entitled “Antibiotics and Processfor Preparation”; and WO 99/64049 A1, published on Dec. 16, 1999, andentitled “Novel Antibacterial Agents.”

Despite such compounds, a need exists for new antibiotics havingimproved properties including, by way of example, increased potencyagainst gram-positive bacteria. In particular, a need exists for newantibiotics which are highly effective against antibiotic-resistantstrains of bacteria, such as methicillin-resistant Staphylococci aureus(MRSA) and methicillin-resistant Staphylococci epidermitis (MRSE).

SUMMARY OF THE INVENTION

The present invention provides novel cross-linkedglycopeptide-cephalosporin compounds which are useful as antibiotics.Among other properties, compounds of this invention have been found topossess surprising and unexpected potency against gram-positive bacteriaincluding methicillin-resistant Staphylococci aureus (MRSA) andmethicillin-resistant Staphylococci epidermitis (MRSE).

Accordingly, in one of its composition aspects, this invention providesa compound of formula I:

or a pharmaceutically-acceptable salt thereof, wherein

X¹ and X² are independently selected from the group consisting ofhydrogen and chloro;

R¹ is —Y^(a)—(W)_(n)—Y^(b)—;

W is selected from the group consisting of —O—, —N(R^(d))—, —S—, —S(O)—,—S(O)₂—, C₃₋₆ cycloalkylene, C₆₋₁₀ arylene and C₂₋₉ heteroarylene;wherein each arylene, cycloalkylene and heteroarylene group isoptionally substituted with 1 to 3 substituents independently selectedfrom R^(b);

Y^(a) and Y^(b) are independently C₁₋₅ alkylene, or when W iscycloalkylene, arylene or heteroarylene, Y^(a) and Y^(b) areindependently selected from the group consisting of a covalent bond andC₁₋₅ alkylene; wherein each alkylene group is optionally substitutedwith 1 to 3 substituents independently selected from —OR^(d),—NR^(d)R^(e), —CO₂R^(d), —C(O)NR^(d)R^(e) and —S(O)₂NR^(d)R^(e);

R² is hydrogen or C₁₋₆ alkyl;

each R³ is independently selected from the group consisting of C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆ cycloalkyl, C₆₋₁₀ aryl, C₂₋₉heteroaryl, C₃₋₆ heterocyclic and R^(a); or two adjacent R³ groups arejoined to form C₃₋₆ alkylene or —O—(C₁₋₆ alkylene)-O—; wherein eachalkyl, alkylene, alkenyl and alkynyl group is optionally substitutedwith 1 to 3 substituents independently selected from the groupconsisting of R^(a) and R^(c); and each aryl, cycloalkyl, heteroaryl andheterocyclic group is optionally substituted with 1 to 3 substituentsindependently selected from the group consisting of R^(b);

one of R⁴ and R⁵ is hydroxy and the other is hydrogen;

R⁶ and R⁷ are independently hydrogen or methyl;

R⁸ is hydrogen or a group of formula (i):

each R^(a) is independently selected from the group consisting of—OR^(d), halo, —SR^(d), —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d),—S(O)₂NR^(d)R^(e), —NR^(d)R^(e), —CO₂R^(d), —OC(O)R^(d),—C(O)NR^(d)R^(e), —NR^(d)C(O)R^(e), —OC(O)NR^(d)R^(e),—NR^(d)C(O)OR^(e), —NR^(d)C(O)NR^(d)R^(e), —CF₃ and —OCF₃;

each R^(b) is independently selected from the group consisting of C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl and R^(a);

each R^(c) is independently selected from the group consisting of C₃₋₆cycloalkyl, C₆₋₁₀ aryl, C₂₋₉ heteroaryl and C₃₋₆ heterocyclic; whereineach cycloalkyl, aryl, heteroaryl and heterocyclic group is optionallysubstituted with 1 to 3 substituents independently selected from thegroup consisting of C₁₋₆ alkyl and R^(f);

each R^(d) and R^(e) is independently selected from the group consistingof hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆ cycloalkyl,C₆₋₁₀ aryl, C₂₋₉ heteroaryl and C₃₋₆ heterocyclic; or R^(d) and R^(e)are joined, together with the atoms to which they are attached, to forma C₃₋₆ heterocyclic ring having 1 to 3 heteroatoms independentlyselected from oxygen, nitrogen or sulfur; wherein each alkyl, alkenyland alkynyl group is optionally substituted with 1 to 3 substituentsindependently selected from the group consisting of R^(c) and R^(f); andeach aryl, cycloalkyl, heteroaryl and heterocyclic group is optionallysubstituted with 1 to 3 substituents independently selected from thegroup consisting of C₁₋₆ alkyl and R^(f);

each R^(f) is independently selected from the group consisting of —OH,—OC₁₋₆ alkyl, —SC₁₋₆ alkyl, —F, —Cl, —NH₂, —NH(C₁₋₆ alkyl), —N(C₁₋₆alkyl)₂, —OC(O)C₁₋₆ alkyl, —C(O)OC₁₋₆ alkyl, —NHC(O)C₁₋₆ alkyl, —C(O)OH,—C(O)NH₂, —C(O)NHC₁₋₆ alkyl, —C(O)N(C₁₋₆ alkyl)₂, —CF₃ and —OCF₃;

m is 0, 1, 2 or 3; and

n is 0 or 1.

This invention is also directed to intermediates useful for preparingcompounds of formula I, and salts thereof. Accordingly, in another ofits composition aspects, this invention provides a compound of formulaII:

or a salt thereof; wherein

R¹ and R² are as defined herein;

P¹ and P² are independently hydrogen or an amino-protecting group;

P³ is hydrogen or a carboxy-protecting group;

Q is a leaving group or a group of the formula:

where R³ and m are as defined herein; and X⁻ is an optionally presentanion; which compounds are useful as intermediates for preparingcompounds of formula I and/or as antibiotics.

In yet another of its composition aspects, this invention provides acompound of formula III:

or salts thereof; wherein R¹, R², P¹ and P² are as defined herein, andP⁴ is hydrogen or a carboxy-protecting group; which compounds are usefulas intermediates for preparing compounds of formula I or II.

In separate and distinct composition aspects, this invention alsoprovides compounds of formulae 2, 5b, 7, 8, 10, 11 and 13 as definedherein, or salts or protected derivatives thereof; which compounds areuseful as intermediates for preparing compounds of formula I and/or asantibiotics.

In another of its composition aspects, this invention provides apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a therapeutically effective amount of a compound of formulaI, or a pharmaceutically-acceptable salt thereof.

While not intending to be limited by theory, the compounds of formula Iare believed to inhibit bacterial cell wall biosynthesis therebyinhibiting the growth of the bacteria or causing lysis of the bacteria.Therefore, among other properties, the compounds of formula I are usefulas antibiotics.

Accordingly, in one of its method aspects, this invention provides amethod of treating a bacterial infection in a mammal, the methodcomprising administering to a mammal a pharmaceutical compositioncomprising a pharmaceutically-acceptable carrier and a therapeuticallyeffective amount of a compound of formula I, or apharmaceutically-acceptable salt thereof.

Additionally, in another of its method aspects, this invention providesa method of inhibiting the growth of bacteria, the method comprisingcontacting bacteria with a growth-inhibiting amount of a compound offormula I, or a pharmaceutically-acceptable salt thereof.

In yet another of its method aspects, this invention provides a methodof inhibiting bacterial cell wall biosynthesis, the method comprisingcontacting bacteria with a cell wall biosynthesis-inhibiting amount of acompound of formula I, or a pharmaceutically-acceptable salt thereof.

This invention is also directed to processes for preparing compounds offormula I or a salt thereof. Accordingly, in another of its methodaspects, this invention provides a process for preparing a compound offormula I, or a salt thereof; the process comprising:

(a) reacting a glycopeptide of formula 1 as defined herein, with acompound of formula 2 as defined herein; or

(b) reacting a compound of formula 10 as defined herein, with a compoundof formula 11 as defined herein; or

(c) reacting a compound of formula 9 as defined herein, with a compoundof formula 13 as defined herein;

to provide a compound of formula I or a salt thereof. In one preferredembodiment, the above process further comprises the step of forming apharmaceutically-acceptable salt of a compound of formula I. Thisinvention is also directed to the product prepared by any of theseprocesses.

This invention is also directed to a compound of formula I, or apharmaceutically-acceptable salt thereof, for use in therapy.Additionally, this invention is directed to the use of a compound offormula I, or a pharmaceutically-acceptable salt thereof, for themanufacture of a medicament for the treatment of a bacterial infectionin a mammal.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides novel glycopeptide—cephalosporin compounds offormula I or pharmaceutically-acceptable salts thereof. These compoundshave multiple chiral centers and, in this regard, the compounds areintended to have the stereochemistry shown. In particular, theglycopeptide portion of the compound is intended to have thestereochemistry of the corresponding naturally-occurring glycopeptide(i.e., vancomycin, chloroorienticin A and the like). The cephalosporinportion of the molecule is intended to have the stereochemistry of knowncephalosporin compounds. However, it will be understood by those skilledin the art that minor amounts of isomers having a differentstereochemistry from that shown may be present in the compositions ofthis invention provided that the utility of the composition as a wholeis not significantly diminished by the presence of such isomers.

Additionally, the linking portion of the compounds of this invention(i.e., R¹) may contain one or more chiral centers. Typically, thisportion of the molecule will be prepared as a racemic mixture. Ifdesired, however, pure stereoisomers (i.e., individual enantiomers ordiastereomers) may be used or a stereoisomer-enriched mixture can beemployed. All such stereoisomers and enriched mixtures are includedwithin the scope of this invention.

In addition, compounds of this invention contain several acidic groups(i.e., carboxylic acid groups) and several basic groups (i.e., primaryand secondary amine groups) and therefore, the compounds of formula Ican exist in various salt forms. All such salt forms are included withinthe scope of this invention. Also, since the compounds of formula Icontain a pyridinium ring, an anionic counterion for the pyridiniumgroup may optionally be present including, but not limited to, halides,such as chloride; carboxylates, such as acetate; and the like.

Furthermore, it will be understood by those skilled in the art thatlabile or chemically unstable compounds which lack any utility due totheir instability are not included within the scope of this invention.For example, it is preferred that compounds of formula I contain atleast two carbon atoms between any oxygen (—O—), nitrogen (—N<) orsulfur (—S—) atoms in the —O—R¹—N(R²)— moiety, since when these atomsare separated by a single carbon atom the resulting compound (i.e.,containing an acetal, hemiacetal, ketal, hemiketal, aminal, hemiamial orthioketal group and the like) may be hydrolytically unstable underacidic conditions.

Preferred Embodiments

In the compounds of formula I, the following substituents and values arepreferred:

In a preferred embodiment, the present invention is directed to,compounds of formula Ia:

or a pharmaceutically-acceptable salt thereof, wherein R¹, R², R³ and mare as defined herein, including preferred embodiments.

In a preferred embodiment, R¹ is —Y^(a)—Y^(b)—, i.e., when n is 0. Inthis embodiment, Y^(a) and Y^(b) are independently C₁₋₅ alkylene groupswherein each alkylene group is optionally substituted with 1 to 3substituents independently selected from —OR^(d), —NR^(d)R^(e),—CO₂R^(d), —C(O)NR^(d)R^(e) and —S(O)₂NR^(d)R^(e) as defined herein.Preferably, Y^(a) and Y^(b) are independently selected from C₁₋₃alkylene; and more preferably, C₁₋₂ alkylene. More preferably, Y^(a) andY^(b) are joined together (i.e., R¹) to form a —(CH₂)₂₋₈— group. Stillmore preferably, Y^(a) and Y^(b) are joined together to form a —(CH₂)₂—,—(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅— or —(CH₂)₆— group. In a particularlypreferred embodiment, Y^(a) and Y^(b) are joined together to form—(CH₂)₃— group.

In another preferred embodiment, R¹ is —Y^(a)—W—Y^(b)—, i.e., when nis 1. In this embodiment, Y^(a) and Y^(b) are independently C₁₋₅alkylene, or when W is cycloalkylene, arylene or heteroarylene, Y^(a)and Y^(b) are independently selected from the group consisting of acovalent bond and C₁₋₅ alkylene. Each alkylene group in this embodimentis optionally substituted with 1 to 3 substituents selected from—OR^(d), —NR^(d)R^(e), —CO₂R^(d), —C(O)NR^(d)R^(e) and —S(O)₂NR^(d)R^(e)as defined herein. When Y^(a) or Y^(b) is an alkylene group, thealkylene group is preferably a C₁₋₃ alkylene group; more preferably, aC₁₋₂ alkylene group; still more preferably, a —(CH₂)₁₋₂— group. In aparticularly preferred embodiment, Y^(a) and Y^(b) are both —CH₂— and Wis C₆₋₁₀ arylene optionally substituted with 1 to 3 substituentsindependently selected from R^(b) as defined herein; more preferably, Wis phenylene. In another preferred embodiment, Y^(a) and Y^(b) are both—CH₂CH₂— and W is —O—.

When present, W is preferably C₆₋₁₀ arylene or —O—. More preferably, Wis phenylene or —O—.

Preferably, R² is hydrogen or C₁₋₃ alkyl. More preferably, R² ishydrogen.

When present, each R³ is preferably independently selected from C₁₋₆alkyl, C₃₋₆ cycloalkyl, —OR^(d), —SR^(d), —F or —Cl; or two adjacent R³groups are joined to form C₃₋₆ alkylene.

In a preferred embodiment, R⁴ is hydroxy and R⁵ is hydrogen. In anotherpreferred embodiment, R⁴ is hydrogen and R⁵ is hydroxy.

Preferably, R⁶ is hydrogen and R⁷ is methyl.

Preferably, R⁸ is hydrogen.

Preferably, one of X¹ and X² is chloro and the other is hydrogen; orboth are chloro. More preferably, X¹ and X² are both chloro.

In a preferred embodiment, R⁴ is hydroxy; R⁵ is hydrogen; R⁶ ishydrogen; R⁷ is methyl; R⁸ is hydrogen; and X¹ and X² are both chloro(i.e., the glycopeptide portion is vancomycin).

In another preferred embodiment, R⁴ is hydrogen; R⁵ is hydroxy; R⁶ ishydrogen; R⁷ is methyl; R⁸ is a group of formula (i); and X¹ and X² areboth chloro (i.e., the glycopeptide portion is chloroorieniticin orA82846B).

In a preferred embodiment, m is 0. In another preferred embodiment, m is1 or 2; more preferably, 1. In still another preferred embodiment, m is2 and the two R³ groups are joined to form a C₃₋₅ alkylene group; morepreferably a C₃₋₄ alkylene group.

A preferred group of compounds of formula I are those of formula Iawherein R¹ is —Y^(a)—(W)_(n)—Y^(b)—, where n is 0 and Y^(a) and Y^(b)are joined together to form a —(CH₂)₂₋₈— group; R² is hydrogen and m is0; or a pharmaceutically-acceptable salt thereof. In this embodiment, R¹(i.e., Y^(a) and Y^(b) taken together) is preferably a —(CH₂)₂—,—(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅— or —(CH₂)₆— group; more preferably,—(CH₂)₃—.

Another preferred group of compounds of formula I are those of formulaIa wherein R¹, R², R³ and m are as defined in Table I, or apharmaceutically-acceptable salt thereof.

TABLE I Ex. R¹ No. Y^(a) W Y^(b) n R² R³ m 1 —CH₂CH₂— — —CH₂— 0 —H — 0 2—CH₂CH₂— — —CH₂— 0 —H 2-CH₃— 1 3 —CH₂CH₂— — —CH₂— 0 —H 3-CH₃— 1 4—CH₂CH₂— — —CH₂— 0 —H 4-CH₃— 1 5 —CH₂CH₂— — —CH₂— 0 —H 2-CH₃O— 1 6—CH₂CH₂— — —CH₂— 0 —H 3-CH₃O— 1 7 —CH₂CH₂— — —CH₂— 0 —H 4-CH₃O— 1 8—CH₂CH₂— — —CH₂— 0 —H 2-CH₃S— 1 9 —CH₂CH₂— — —CH₂— 0 —H 3-CH₃S— 1 10—CH₂CH₂— — —CH₂— 0 —H 4-CH₃S— 1 11 —CH₂CH₂— — —CH₂— 0 —H 2-F— 1 12—CH₂CH₂— — —CH₂— 0 —H 3-F— 1 13 —CH₂CH₂— — —CH₂— 0 —H 4-F— 1 14 —CH₂CH₂—— —CH₂— 0 —H 2-Cl— 1 15 —CH₂CH₂— — —CH₂— 0 —H 3-Cl— 1 16 —CH₂CH₂— ——CH₂— 0 —H 4-Cl— 1 17 —CH₂CH₂— — —CH₂— 0 —H 2-Ph-¹ 1 18 —CH₂CH₂— — —CH₂—0 —H 3-Ph- 1 19 —CH₂CH₂— — —CH₂— 0 —H 4-Ph- 1 20 —CH₂CH₂— — —CH₂— 0 —H4-cyclopropyl- 1 21 —CH₂CH₂— — —CH₂— 0 —H 4-[HOOCCH₂S—] 1 22 —CH₂CH₂— ——CH₂— 0 —H 4-NH₂C(O)— 1 23 —CH₂CH₂— — —CH₂— 0 —H 2,3-di-CH₃— 2 24—CH₂CH₂— — —CH₂— 0 —H 3,4-di-CH₃— 2 25 —CH₂CH₂— — —CH₂— 0 —H 3,5-di-CH₃—2 26 —CH₂CH₂— — —CH₂— 0 —H 3,4-di-CH₃O— 2 27 —CH₂CH₂— — —CH₂— 0 —H3-CH₃-4-CH₃O— 2 28 —CH₂CH₂— — —CH₂— 0 —H 3-CH₃O-4-F— 2 29 —CH₂CH₂— ——CH₂— 0 —H 2,3-[—(CH₂)₄—) 2 30 —CH₂CH₂— — —CH₂— 0 —H 2,3-(—CH₂CH₂CH₂—) 231 —(CH₂)₃— — —(CH₂)₃— 0 —H — 0 32 —CH₂CH₂— —O— —CH₂CH₂— 1 —H — 0 33—CH₂— 1,4-(-Ph-)² —CH₂— 1 —H — 0 ¹Ph = phenyl ²1,4-(-Ph-) =1,4-phenylene

In the intermediate of formula II:

Q is preferably halo or the defined pyridinium group.

P¹ is preferably hydrogen or tert-butoxycarbonyl.

P² is preferably hydrogen or triphenylmethyl.

P³ is preferably hydrogen or p-methoxybenzyl.

R¹, R², R³ and m are preferably as defined herein including anypreferred embodiments, substituents or values.

In the intermediate of formula III:

P¹ is preferably hydrogen or tert-butoxycarbonyl.

P² is preferably hydrogen, formyl or triphenylmethyl.

P⁴ is preferably hydrogen, C₁₋₄ alkyl or p-methoxybenzyl.

R¹ and R² are preferably as defined herein including any preferredembodiments, substituents or values

Definitions

When describing the compounds, compositions, methods and processes ofthis invention, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” refers to a monovalent saturated hydrocarbon groupwhich may be linear or branched. Unless otherwise defined, such alkylgroups typically contain from 1 to 10 carbon atoms. Representative alkylgroups include, by way of example, methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl,n-octyl, n-nonyl, n-decyl and the like.

The term “alkylene” refers to a divalent saturated hydrocarbon groupwhich may be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like.

The term “alkenyl” refers to a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to 10 carbonatoms. Representative alkenyl groups include, by way of example,ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and thelike.

The term “alkynyl” refers to a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbonatoms. Representative alkynyl groups include, by way of example,ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like.

The term “aryl” refers to a monovalent aromatic hydrocarbon having asingle ring (i.e., phenyl) or fused rings (i.e., naphthalene). Unlessotherwise defined, such aryl groups typically contain from 6 to 10carbon ring atoms. Representative aryl groups include, by way ofexample, phenyl and naphthalene-1-yl, naphthalene-2-yl, and the like.

The term “arylene” refers to a divalent aromatic hydrocarbon having asingle ring (i.e., phenylene) or fused rings (i.e., naphthalenediyl).Unless otherwise defined, such arylene groups typically contain from 6to 10 carbon ring atoms. Representative arylene groups include, by wayof example, 1,2-phenylene, 1,3-phenylene, 1,4-phenylene,naphthalene-1,5-diyl, naphthalene-2,7-diyl, and the like.

The term “cycloalkyl” refers to a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms. Representative cycloalkylgroups include, by way of example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and the like.

The term “cycloalkylene” refers to a divalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkylene groupstypically contain from 3 to 10 carbon atoms. Representativecycloalkylene groups include, by way of example, cyclopropane-1,2-diyl,cyclobutyl-1,2-diyl, cyclobutyl-1,3-diyl, cyclopentyl-1,2-diyl,cyclopentyl-1,3-diyl, cyclohexyl-1,2-diyl, cyclohexyl-1,3-diyl,cyclohexyl-1,4-diyl, and the like.

The term “halo” refers to fluoro, chloro, bromo and iodo.

The term “heteroaryl” refers to a monovalent aromatic group having asingle ring or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms. Representative heteroaryl groupsinclude, by way of example, monovalent species of pyrrole, imidazole,thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole,isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine,indole, benzofuran, benzothiophene, benzimidazole, benzthiazole,quinoline, isoquinoline, quinazoline, quinoxaline and the like, wherethe point of attachment is at any available carbon or nitrogen ringatom.

The term “heteroarylene” refers to a divalent aromatic group having asingle ring or two fused rings and containing at least one heteroatom(typically 1 to 3 heteroatoms) selected from nitrogen, oxygen or sulfurin the ring. Unless otherwise defined, such heteroarylene groupstypically contain from 5 to 10 total ring atoms. Representativeheteroarylene groups include, by way of example, divalent species ofpyrrole, imidazole, thiazole, oxazole, furan thiophene, triazole,pyrazole, isoxazole, isothiazole, pyridine, pyrazine, pyridazine,pyrimidine, triazine, indole, benzofuran, benzothiophene, benzimidazole,benzthiazole, quinoline, isoquinoline, quinazoline, quinoxaline and thelike, where the point of attachment is at any available carbon ornitrogen ring atom.

The term “heterocyclyl” or “heterocyclic” refers to a monovalentsaturated or unsaturated (non-aromatic) group having a single ring ormultiple condensed rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heterocyclic groups typicallycontain from 2 to 9 total ring atoms. Representative heterocyclic groupsinclude, by way of example, monovalent species of pyrrolidine,imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, morpholine,thiomorpholine, piperazine, 3-pyrroline and the like, where the point ofattachment is at any available carbon or nitrogen ring atom.

The term “cephalosporin” is used herein in its art recognized manner torefer to a β-lactam ring system having the following general formula andnumbering system:

The term “glycopeptide antibiotic” or “glycopeptide” is used herein inits art recognized manner to refer to the class of antibiotics known asglycopeptides or dalbahpeptides. See, for example, R. Nagarajan,“Glycopeptide Anitibiotics”, Marcel Dekker, Inc. (1994) and referencescited therein. Representative glycopeptides include vancomycin, A82846A(eremomycin), A82846B (chloroorienticin A), A82846C, PA-42867-A(orienticin A), PA-42867-C, PA-42867-D and the like.

The term “vancomycin” is used herein in its art recognized manner torefer to the glycopeptide antibiotic known as vancomycin. In thecompounds of the present invention, the point of attachment for thelinking moiety is at the “C-terminus” of vancomycin.

The term “pharmaceutically-acceptable salt” refers to a salt which isacceptable for administration to a patient, such as a mammal (e.g.,salts having acceptable mammalian safety for a given dosage regime).Such salts can be derived from pharmaceutically-acceptable inorganic ororganic bases and from pharmaceutically-acceptable inorganic or organicacids. Salts derived from pharmaceutically-acceptable inorganic basesinclude aluminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic, manganous, potassium, sodium, zinc and the like.Particularly preferred are ammonium, calcium, magnesium, potassium andsodium salts. Salts derived from pharmaceutically-acceptable organicbases include salts of primary, secondary and tertiary amines, includingsubstituted amines, cyclic amines, naturally-occurring amines and thelike, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically-acceptable acidsinclude acetic, ascorbic, benzenesulfonic, benzoic, camphosulfonic,citric, ethanesulfonic, fumaric, gluconic, glucoronic, glutamic,hippuric, hydrobromic, hydrochloric, isethionic, lactic, lactobionic,maleic, malic, mandelic, methanesulfonic, mucic, naphthalenesulfonic,nicotinic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric,tartaric, p-toluenesulfonic and the like. Particularly preferred arecitric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric andtartaric acids.

The term “salt thereof” refers to a compound formed when the hydrogen ofan acid is replaced by a cation, such as a metal cation or an organiccation and the like (e.g., an NH₄ ⁺ cation and the like). Preferably,the salt is a pharmaceutically-acceptable salt, although this is notrequired for salts of intermediate compounds which are not intended foradministration to a patient.

The term “therapeutically effective amount” refers to an amountsufficient to effect treatment when administered to a patient in need oftreatment.

The term “treating” or “treatment” as used herein refers to the treatingor treatment of a disease or medical condition (such as a bacterialinfection) in a patient, such as a mammal (particularly a human or acompanion animal) which includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient.

The term “growth-inhibiting amount” refers to an amount sufficient toinhibit the growth or reproduction of a microorganism or sufficient tocause death or lysis of the microorganism including gram-positivebacteria.

The term “cell wall biosynthesis-inhibiting amount” refers to an amountsufficient to inhibit cell wall biosynthesis in a microorganismincluding gram-positive bacteria.

The term “leaving group” refers to a functional group or atom which canbe displaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; and sulfonic ester groups, such as mesylate, tosylate,brosylate, nosylate and the like; activated ester groups, such as suchas 7-azabenzotriazole-1-oxy and the like; acyloxy groups, such asacetoxy, trifluoroacetoxy and the like.

The term “protected derivatives thereof” refers to a derivative of thespecified compound in which one or more functional groups of thecompound are protected from undesired reactions with a protecting orblocking group. Functional groups which may be protected include, by wayof example, carboxylic acid groups, amino groups, hydroxyl groups, thiolgroups, carbonyl groups and the like. Representative protecting groupsfor carboxylic acids include esters (such as a p-methoxybenzyl ester),amides and hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters;for thiol groups, thioethers and thioesters; for carbonyl groups,acetals and ketals; and the like. Such protecting groups are well-knownto those skilled in the art and are described, for example, in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, New York, 1999, and references cited therein.

The term “amino-protecting group” refers to a protecting group suitablefor preventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), and the like.

The term “carboxy-protecting group” refers to a protecting groupsuitable for preventing undesired reactions at an carboxy group.Representative carboxy-protecting groups include, but are not limitedto, esters, such as methyl, ethyl, tert-butyl, benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), diphenylmethyl (benzhydryl, DPM) and thelike.

General Synthetic Procedures

The cross-linked glycopeptide-cephalosporin compounds of this inventioncan be prepared from readily available starting materials using thefollowing general methods and procedures. It will be appreciated thatwhere typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can be readilydetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions for protection and deprotection of suchfunctional groups are well known in the art. Protecting groups otherthan those illustrated in the procedures described herein may be used,if desired. For example, numerous protecting groups, and theirintroduction and removal, are described in T. W. Greene and G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York,1999, and references cited therein.

In a preferred method of synthesis, the compounds of formula I areprepared by reacting a glycopeptide of formula 1:

wherein R⁴, R⁵, R⁶, R⁷, R⁸, X¹ and X² are as defined herein, or a saltthereof, with a compound of formula 2:

wherein R¹, R², R³ and m are as defined herein, or a salt or protectedderivative thereof, to provide a compound of formula I, or a salt orprotected derivative thereof.

Typically, this reaction is conducted by coupling glycopeptide 1, or asalt thereof, with about 0.5 to about 1.5 equivalents, preferably about0.9 to about 1.1 equivalents, of a compound of formula 2 in an inertdiluent, such as DMF, using a conventional carboxylic acid-amine(peptide) coupling reagent. In this reaction, glycopeptide 1, or a saltthereof, is typically first contacted with the coupling reagent in thepresence of an excess, preferably about 1.8 to about 2.2 equivalents, ofan amine, such as diisopropylethylamine at a temperature ranging fromabout −20° C. to about 25° C., preferably at ambient temperature, forabout 0.25 to about 3 hours. Preferably, excess trifluoroacetic acid(typically about 2 equivalents) is then added to form a TFA salt of anyexcess diisopropylethylamine. The reaction is then generally cooled to atemperature of about −20° C. to about 10° C., preferably to about 0° C.,and intermediate 2 is added, followed by excess 2,4,6-collidine. Thisreaction is typically maintained at about 0° C. for about 1 to about 6hours, or until the reaction is substantially complete.

A preferred coupling reagent for use in this reaction comprises about0.5 to about 1.5 equivalents, preferably about 0.9 to about 1.1equivalents, of benzotriazol-1-yloxytripyrrolidinophosphoniumhexafluorophosphate (PyBOP) and about 0.5 to about 1.5 equivalents,preferably about 0.9 to about 1.1 equivalents, of 1-hydroxybenzotriazole(HOBT) or 1-hydroxy-7-azabenzotriazole (HOAT). Other suitable couplingreagents include O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU); bis(2-oxo-3-oxazolidinyl)phosphinic chloride(BOP—Cl); diphenylphosphoryl azide (DPPA); diphenylphosphinic chloride;diphenyl chlorophosphate (DPCP) and HOAT; pentafluorophenyldiphenylphosphinate and the like.

After the coupling reaction is complete, any protecting groups presentin the product are then removed using conventional procedures andreagents. Upon completion of this reaction, the reaction product, i.e.,a compound of formula I, is isolated and purified using conventionalprocedures, such as column chromatography, HPLC, recrystallization andthe like.

Glycopeptides of formula 1 suitable for use in the above procedure areeither commercially available or they can be prepared by fermentation ofthe appropriate glycopeptide-producing organism, followed by isolationof the glycopeptide from the resulting fermentation broth using artrecognized procedures and equipment.

The cephalosporin intermediate 2 used in the above procedure is readilyprepared from commercially available starting materials and reagentsusing conventional procedures. By way of example, intermediate 2 can beprepared as shown in Scheme A:

As illustrated in Scheme A, thiazole intermediate 3 (wherein R⁹ is anamino-protecting group, such as a trityl group, and R¹⁰ is acarboxy-protecting group, such as an ethyl group) is first reacted withan ω-functionalized amine of formula 4 (wherein R¹ and R² are as definedherein, R¹¹ is an amino-protecting group, such as a tert-butoxycarbonyl(BOC) group, and Z¹ is a leaving group, such as chloro, bromo, iodo,meslyate, tosylate and the like) to provide, after removal of thecarboxy-protecting group (i.e., R¹⁰), an intermediate of formula 5a.

This reaction is typically conducted by first contacting 3 with about1.0 to about 1.1 equivalents, preferably with about 1.02 to about 1.06equivalents, of a compound of formula 4 in an inert diluent, such asDMF, at a temperature ranging from about 0° C. to about 50° C.,preferably at ambient temperature, for about 0.5 to about 6 hours, oruntil the reaction is substantially complete. This reaction is typicallyconducted in the presence of excess, preferably about 1.1 to about 5equivalents, of a base, such as cesium carbonate. Additionally, when Z¹is chloro or bromo, a catalytic amount, preferably about 0.2 to about0.5 equivalents, of an trialkylammonium iodide, such astetrabutylammonium iodide, is optionally added to facilitate thereaction by generating the iodo derivative of 4 in situ.

Removal of the carboxy-protecting group (i.e., R¹⁰) then affordsintermediate 5a. For example, when the carboxy-protecting group is analkyl ester, such as an ethyl group, the ester is readily hydrolyzed tothe carboxylic acid by contacting the ester with an excess, preferablywith about 1.1 to about 2.5 equivalents, of an alkali metal hydroxide,such as sodium hydroxide or potassium hydroxide. This reaction istypically conducted in an inert diluent, such as ethanol, at atemperature ranging from about 0° C. to about 100° C. for about 0.5 toabout 6 hours, or until the reaction is substantially complete, toafford intermediate 5a.

Thiazole compounds of formula 3 are commercially available from, forexample, Aldrich, Milwaukee, Wis., or can be prepared from commerciallyavailable starting materials and reagents using conventional procedures.

Similarly, ω-functionalized amines of formula 4 are readily preparedfrom commercially-available starting materials and reagents usingconventional procedures. Preferred compounds of formula 4 include, byway of illustration, N—BOC-3-bromopropylamine; N—BOC-6-iodohexylamine;N—BOC-2-(2-iodoethoxy)-ethylamine; N—BOC-4-(iodomethyl)benzylamine; andthe like. These compounds are readily prepared from commerciallyavailable starting using well-known reagents and reaction conditions.

Intermediate 5a is then chlorinated to provide intermediate 5b. Thisreaction is typically conducted by contacting 5a with about 1.0 to about1.2 equivalents, of a chlorinating agent, such as N-chlorosuccinimide,in an inert diluent, such as chloroform or DMF, at ambient temperaturefor about 6 to about 24 hours, or until the reaction is substantiallycomplete.

5-Chloro-1,3-thiazole intermediate 5b is then coupled with intermediate6 (wherein R¹² is hydrogen or a suitable carboxyl protecting group, suchas a p-methoxybenzyl group) to provide intermediate 7. When R¹² isp-methoxybenzyl, intermediate 6 is commercially available from Otsuka,Japan. Typically, this reaction is conducted by contacting 5b with about0.8 to about 1 equivalents of 6 in the presence of a coupling reagentunder conventional coupling reaction conditions. A preferred couplingreagent for this reaction is phosphorous oxychloride (typically about1.1 to about 1.2 equivalents) and an excess amount of an amine, such as2,4,6-collidine or diisopropylethylamine. The coupling reaction istypically conducted in an inert diluent, such as THF, at a temperatureranging from about −50° C. to about 25° C. for about 0.5 to about 6hours, or until the reaction is substantially complete, to affordintermediate 7. To avoid isomerization, this reaction is preferablyconducted at −35° C. using 2,4,6-collidine as the base.

Intermediate 7 is then reacted with a pyridine or substituted pyridineto afford intermediate 8, where R³ and n are as defined herein. Thisreaction is typically conducted by first exchanging the chloro group in7 with an iodo group by contacting 7 with about one equivalent of sodiumiodide in acetone (Finkelstein reaction) or DMF at ambient temperaturefor about 0.25 to about 2 hours. The resulting iodo intermediate istypically not isolated, but is reacted in situ with about 1.1 to about1.6 equivalents of a pyridine or substituted pyridine to afford 8.Typically, this reaction is conducted at ambient temperature for about 1to about 12 hours, or until the reaction is substantially complete. Thepyridine or substituted pyridines used in this reaction are eithercommercially available or can be prepared from commercially availablestarting materials and reagents using conventional procedures.Representative pyridine derivatives for use in this reaction includepyridine, 2-picoline, 3-picoline, 4-picoline, 2-methoxypyridine,3-methoxypyridine, 4-methoxypyridine, 2-thiomethoxypyridine,3-thiomethoxypyridine, 4-thiomethoxypyridine,4-carboxythiomethoxypyridine, 2-fluoropyridine, 3-fluoropyridine,4-fluoropyridine, 2-chloropyridine, 3-chloropyridine, 4-chloropyridine,2-phenylpyridine, 3-phenylpyridine, 4-phenylpyridine,4-cyclopropylpyridine, nicotinic acid, isonicotinic acid, nicotinamide,isonicotinamide, 2,3-lutidine, 3,4-lutidine, 3,5-lutidine,3,4-dimethoxypyridine, 4-methoxy-3-methylpyridine,4-fluoro-3-methoxypyridine, 2,3-cyclopentenopyridine,2,3-cyclohexenopyridine and the like.

Alternatively, intermediate 5b can be coupled with a compound of formula9:

wherein R³, R¹² and m are as defined herein, to afford intermediate 8.This reaction is typically conducted by contacting 5b with about 0.9 toabout 1.1 equivalents of intermediate 9, or a salt thereof, in an inertdiluent, such as DMF, in the presence of a coupling reagent, such as1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC); PyBOP and HOAT orHOBT; HATU; BOP—Cl; DPPA; DPCP and HOAT; and the like. Generally, thecoupling reaction is conducted at a temperature ranging from about −40°C. to about 25° C. for about 1 to about 12 hours, or until the reactionis substantially complete. Compounds of formula 9 are readily preparedfrom intermediate 6 by reaction of 6 with pyridine or a substitutedpyridine under reaction conditions similar to those described above.

Removal of the protecting groups from intermediate 8 using conventionalprocedures and reagents then affords cephalosporin intermediate 2. Forexample, when R⁹ is trityl, R¹¹ is tert-butoxycarbonyl and R¹² ispara-methoxybenzyl, the protecting groups are conveniently removed bytreating 8 with excess trifluoroacetic acid and excess anisole ortriethylsilane in an inert diluent, such as dichloromethane or heptane,at ambient temperature for about 1 to about 12 hours, or until thereaction is complete. The resulting deprotected cephalosporin 2 istypically isolated and purified using conventional procedures, such asprecipitation, lyophilzation and reverse-phase HPLC.

Alternatively, compounds of formula I can be prepared by reacting aglycopeptide derivative of formula 10:

or a salt thereof, wherein R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, X¹ and X² are asdefined herein, with a cephalosporin derivative of formula 11:

or a salt or protected derivative thereof (wherein R³ and m are asdefined herein) to afford a compound of formula I, or salt thereof.

This reaction is typically conducted by contacting 10 with about 1 toabout 1.5 equivalents of 11 in an inert diluent, such as water, methanolor mixtures thereof, at a pH ranging from about 4 to about 6.5. Thisreaction is generally conducted at a temperature ranging from about −20°C. to about 40° C. for about 1 to about 6 hours, or until the reactionis substantially complete.

The vancomycin derivatives of formula 10 employed in this reaction arereadily prepared by coupling vancomycin, or a salt thereof, with aphthalimido derivative of the formula:

wherein R¹ is as defined herein, under conventional coupling conditions.For example, this reaction is typically conducted by contactingvancomycin with about 1.1 to about 1.2 equivalents of the phthalimidocompound in the presence of a coupling reagent, such as PyBOP and HOATand the like, and a suitable base, such as diisopropylethylamine.Generally, the reaction is conducted in an inert diluent, such as DMF,at a temperature ranging from about −20° C. to about 40° C. for about0.5 to about 6 hours, or until the reaction is substantially complete.The phthalimido compounds employed in this reaction are readily preparedusing conventional procedures and reagents.

Cephalosporin derivatives of formula 11 can be prepared, for example, bycoupling a compound of formula 9 above with a thiazole compound offormula 12:

or a salt thereof, wherein R¹³ is hydrogen or an amino-protecting group(such as a formyl or trityl group). This coupling reaction is typicallyconducted by contacting 9 with about 0.9 to about 1.1 equivalents of 12in an inert diluent, such as DMF, in the presence of a coupling reagent,such as EDC and HOAT, and a suitable base, such as 2,4,6-collidine.Generally, this reaction is conducted at a temperature ranging fromabout −20° C. to about 20° C. for about 0.5 to about 6 hours, or untilthe reaction is substantially complete.

Additionally, compounds of formula I can be prepared by reacting aglycopeptide derivative of formula 13:

or a salt thereof, wherein R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, X¹ and X² are asdefined herein, with a compound of formula 9 above to afford a compoundof formula I, or a salt thereof.

This coupling reaction is typically conducted by contacting 13 with acoupling reagent, such as DIPC and HOAT, and about 0.5 to about 2equivalents of 9 in an inert diluent, such as DMF, in the presence of asuitable base, such as 2,4,6-collidine. Generally, this reaction isconducted at a temperature ranging from about −20° C. to about 40° C.for about 1 to about 6 hours, or until the reaction is substantiallycomplete.

The compound of formula 13 used in this reaction is readily prepared bycoupling vancomycin with intermediate 5b above, using conventionalcoupling procedures described herein.

Further details regarding specific reaction conditions and proceduresfor preparing representative compounds of this invention orintermediates thereto are described in the Examples set forth below.

Pharmaceutical Formulations

The cross-linked glycopeptide-cephalosporin compounds of this inventionare typically administered to a patient in the form of a pharmaceuticalcomposition. Accordingly, in one of its composition aspects, thisinvention is directed to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a therapeuticallyeffective amount of a compound of formula I or a pharmaceuticallyacceptable salt thereof.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of this invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof bacterial infection. In this regard, the preparation of a suitablepharmaceutical composition for a particular mode of administration, suchas oral, topical, inhaled or parenteral administration, is well withinthe scope of those skilled in the pharmaceutical arts. Additionally, theingredients for such compositions are commercially-available from, forexample, Sigma, P.O. Box 14508, St. Louis, Mo. 63178. By way of furtherillustration, conventional formulation techniques are described inRemington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia,Pa. 17^(th) Ed. (1985) and “Modern Pharmaceutics,” Marcel Dekker, Inc.3^(rd) Ed. (G. S. Banker & C. T. Rhodes, Eds.).

The pharmaceutical compositions of this invention will typically containa therapeutically effective amount of a compound of formula I or apharmaceutically-acceptable salt thereof. Typically, such pharmaceuticalcompositions will contain from about 0.1 to about 90% by weight of theactive agent, and more generally from about 10 to about 30% of theactive agent.

Preferred pharmaceutical compositions of this invention are thosesuitable for parenteral administration, particularly intravenousadministration. Such pharmaceutical compositions typically comprise asterile, physiologically-acceptable aqueous solution containing atherapeutically effective amount of a compound of formula I or apharmaceutically-acceptable salt thereof.

Physiologically-acceptable aqueous carrier solutions suitable forintravenous administration of active agents are well-known in the art.Such aqueous solutions include, by way of example, 5% dextrose, Ringer'ssolutions (lactated Ringer's injection, lactated Ringer's plus 5%dextrose injection, acylated Ringer's injection), Normosol-M, Isolyte E,and the like.

Optionally, such aqueous solutions may contain a co-solvent, forexample, polyethylene glycol; a chelating agent, for example,ethylenediamine tetracetic acid; a solubilizing agent, for example, acyclodextrin; an anti-oxidant, for example, sodium metabisulphite; andthe like.

If desired, the aqueous pharmaceutical compositions of this inventioncan be lyophilized and subsequently reconstituted with a suitablecarrier prior to administration. In a preferred embodiment, thepharmaceutical composition is a lyophilized composition comprising apharmaceutically-acceptable carrier and a therapeutically effectiveamount of a compound of formula I, or a pharmaceutically-acceptable saltthereof. Preferably, the carrier in this composition comprises sucrose,mannitol, dextrose, dextran, lactose or a combination thereof. Morepreferably, the carrier comprises sucrose, mannitol, or a combinationthereof.

In one embodiment, the pharmaceutical compositions of this inventioncontain a cyclodextrin. When used in the pharmaceutical compositions ofthis invention, the cyclodextrin is preferablyhydroxypropyl-β-cyclodextrin or sulfobutyl ether β-cyclodextrin. In suchformulations, the cyclodextrin will comprise about 1 to 25 weightpercent; preferably, about 2 to 10 weight percent of the formulation.Additionally, the weight ratio of cyclodextrin to active agent willtypically range from about 1:1 to about 10:1.

The pharmaceutical compositions of this invention are preferablypackaged in a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be packaged in sterile, hermetically-sealed ampoules and the like.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

Formulation Example A

A frozen solution suitable for preparing an injectable solution isprepared as follows:

Ingredients Amount Active Compound 10 to 1000 mg Excipients (e.g.,dextrose) 0 to 50 g Water for Injection Solution 10 to 100 mL

-   -   Representative Procedure: The excipients, if any, are dissolved        in about 80% of the water for injection and the active compound        is added and dissolved. The pH is adjusted with 1 M sodium        hydroxide to 3 to 4.5 and the volume is then adjusted to 95% of        the final volume with water for injection. The pH is checked and        adjusted, if necessary, and the volume is adjusted to the final        volume with water for injection. The formulation is then sterile        filtered through a 0.22 micron filter and placed into a sterile        vial under aseptic conditions. The vial is capped, labeled and        stored frozen.

Formulation Example B

A lyophilized powder suitable for preparing an injectable solution isprepared as follows:

Ingredients Amount Active Compound 10 to 1000 mg Excipients (e.g.,mannitol and/or sucrose) 0 to 50 g Buffer Agent (e.g., citrate) 0 to 500mg Water for Injection 10 to 100 mL

-   -   Representative Procedure: The excipients and/or buffering        agents, if any, are dissolved in about 60% of the water for        injection. The active compound is added and dissolved and the pH        is adjusted with 1 M sodium hydroxide to 3 to 4.5 and the volume        is adjusted to 95% of the final volume with water for injection.        The pH is checked and adjusted, if necessary, and the volume is        adjusted to the final volume with water for injection. The        formulation is then sterile filtered through a 0.22 micron        filter and placed into a sterile vial under aseptic conditions.        The formulation is then freeze-dried using an appropriate        lyophilization cycle. The vial is capped (optionally under        partial vacuum or dry nitrogen), labeled and stored under        refrigeration.

Formulation Example C

An injectable solution for intravenous administration to a patient isprepared from Formulation Example B above as follows:

-   -   Representative Procedure: The lyophilized powder of Formulation        Example B (e.g., containing 10 to 1000 mg of active compound) is        reconstituted with 20 mL of sterile water and the resulting        solution is further diluted with 80 mL of sterile saline in a        100 mL infusion bag. The diluted solution is then administered        to the patient intravenously over 30 to 120 minutes.        Utility

The cross-linked glycopeptide-cephalosporin compounds of the inventionare useful as antibiotics. For example, the compounds of this inventionare useful for treating or preventing bacterial infections and otherbacteria-related medical conditions in mammals, including humans andtheir companion animals (i.e., dogs, cats, etc.) which are caused bymicroorganisms susceptible to the compounds of this invention.

Accordingly, in one of its method aspects, this invention provides amethod of treating a bacterial infection in a mammal, the methodcomprising administering to a mammal in need of treatment, apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a therapeutically effective amount of a compound of formulaI, or a pharmaceutically-acceptable salt thereof.

By way of illustration, the compounds of this invention are particularlyuseful for treating or preventing infections caused by Gram-positivebacteria and related microorganisms. For example, the compounds of thisinvention are effective for treating or preventing infections caused bycertain Enterococcus spp.; Staphylococcus spp., including coagulasenegative staphylococci (CNS); Streptococcus spp.; Listeria spp.;Clostridium ssp.; Bacillus spp.; and the like. Examples of bacterialspecies effectively treated with the compounds of this inventioninclude, but are not limited to, methicillin-resistant Staphylococcusaureus (MRSA); methicillin-susceptible Staphylococcus aureus (MSSA);glycopeptide intermediate-susceptible Staphylococcus aureus (GISA);methicillin-resistant Staphylococcus epidermitis (MRSE);methicillin-sensitive Staphylococcus epidermitis (MSSE);vancomycin-sensitive Enterococcus faecalis (EFSVS); vancomycin-sensitiveEnterococcus faecium (EFMVS); penicillin-resistant Streptococcuspneumoniae (PRSP); Streptococcus pyogenes; and the like. Compounds ofthis invention are less effective or not effective for treating orpreventing infections caused by strains of bacteria which are resistantto both vancomycin and cephalosporins.

Representative types of infections or bacteria-related medicalconditions which can be treated or prevented with the compounds of thisinvention include, but are not limited to, skin and skin structureinfections, urinary tract infections, pneumonia, endocarditis,catheter-related blood stream infections, osteomyelitis, and the like.In treating such conditions, the patient may already be infected withthe microorganism to be treated or merely be susceptible to infection inwhich case the active agent is administered prophylactically.

The compounds of this invention are typically administered in atherapeutically effective amount by any acceptable route ofadministration. Preferably, the compounds are administered parenterally.The compounds may be administered in a single daily dose or in multipledoses per day. The treatment regimen may require administration overextended periods of time, for example, for several days or for one tosix weeks or longer. The amount of active agent administered per dose orthe total amount administered will typically be determined by thepatient's physician and will depend on such factors as the nature andseverity of the infection, the age and general health of the patient,the tolerance of the patient to the active agent, the microorganism(s)causing the infection, the route of administration and the like.

In general, suitable doses will range of from about 0.25 to about 10.0mg/kg/day of active agent, preferably from about 0.5 to about 2mg/kg/day. For an average 70 kg human, this would amount to about 15 toabout 700 mg per day of active agent, or preferably about 35 to about150 mg per day.

Additionally, the compounds of this invention are effective forinhibiting the growth of bacteria. In this embodiment, bacteria arecontacted either in vitro or in vivo with a growth-inhibiting amount ofa compound of formula I or pharmaceutically-acceptable salt thereof.Typically, a growth-inhibiting amount will range from about 0.008 μg/mLto about 50 μg/mL; preferably from about 0.008 μg/mL to about 25 μg/mL;and more preferably, from about 0.008 μg/mL to about 10 μg/mL.Inhibition of bacterial growth is typically evidenced by a decrease orlack of reproduction by the bacteria and/or by lysis of the bacteria,i.e., by a decrease in colony-forming units in a given volume (i.e., permL) over a given period of time (i.e., per hour) compared to untreatedbacteria.

The compounds of this invention are also effective for inhibiting cellwall biosynthesis in bacteria. In this embodiment, bacterial arecontacted either in vitro or in vivo with a cell wallbiosynthesis-inhibiting amount of a compound of formula I orpharmaceutically-acceptable salt thereof. Typically, a cell wallbiosynthesis-inhibiting amount will range from about 0.04 μg/mL to about50 μg/mL; preferably from about 0.04 μg/mL to about 25 μg/mL; and morepreferably, from about 0.04 μg/mL to about 10 μg/mL. Inhibition of cellwall biosynthesis in bacteria is typically evidenced by inhibition orlack of growth of the bacteria including lysis of the bacteria.

In addition to surprising and unexpected antibacterial properties,compounds of this invention have also been found to possess acceptablemammalian safety and acceptable aqueous solubility. Additionally,compounds of this invention have been found to have surprising andunexpectedly rapid cidality against certain bacteria, includingmethicillin-resistant Staphylococci aureus (MRSA) andmethicillin-resistant Staphylococci epidermitis (MRSE). Theseproperties, as well as the antibiotic utility of the compounds of thisinvention, can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art. For example, representativeassays are described in further detail in the following Examples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeaning.

-   -   BOC=tert-butoxycarbonyl    -   CFU=colony-forming units    -   DCM=dichloromethane    -   DIPEA diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   DMSO=dimethyl sulfoxide    -   EtOAc=ethyl acetate    -   HOAT=1-hydroxy-7-azabenzotriazole    -   HPLC=high performance liquid chromatography    -   MIC=minimum inhibitory concentration    -   MS=mass spectrometry    -   PMB=p-methoxybenzyl    -   PyBOP=benzotriazol-1-yloxytripyrrolidino-phosphonium        hexafluorophosphate    -   THF=tetrahydrofuran    -   TLC=thin layer chromatography    -   TFA=trifluoroacetic acid

All temperatures reported in the following examples are in degreesCelsius (° C.) unless otherwise indicated. Also, unless noted otherwise,reagents, starting materials and solvents were purchased from commercialsuppliers (such as Aldrich, Fluka, Sigma and the like) and were usedwithout further purification. Vancomycin hydrochloride semi-hydrate waspurchased from Alpharma, Inc., Fort Lee, N.J. 07024 (Alpharma AS, Oslo,Norway).

Reverse-phase HPLC was typically conducted using a C₁₈ column and (A)98% water, 2% acetonitrile, 0.1% TFA, with an excessing gradient (e.g.,0 to about 70%) of (B) 10% water, 90% acetonitrile, 0.1% TFA, unlessotherwise stated.

Example A Synthesis of(7R)-7-[(Z)-2-(2-Amino-5-chlorothiazol-4-yl)-2-(3-aminopropoxyimino)acetamido]-3-[(1-pyridinio)methyl]-3-cephem-4-carboxylatebis-Trifluoroacetic Acid Salt

The following synthesis is illustrated, in part, in Scheme A above.

Step 1—Preparation of N-(tert-Butoxycarbonyl)-3-bromopropylamine (i.e.Compound 4 where R¹ is —(CH₂)₃—, R² is hydrogen R¹¹ is BOC, and Z¹ isbromo)

3-Bromopropylamine hydrobromide (100 g, 457 mmol) was suspended in 1.6 Lof anhydrous THF. This mixture was cooled to 0° C. in an ice/water bathand stirred vigorously while 190 mL of triethylamine was added. To thismixture was added dropwise tert-butoxycarbonyl anhydride (112.6 g, 516mmol) in 200 mL THF. The ice bath was allowed to warm to ambienttemperature and the mixture was stirred overnight at which time TLCindicated the reaction was complete. The mixture was then filtered andthe filtrate was concentrated under vacuum. The residual oil was dilutedwith 1500 mL hexane and stored at −20° C. for 3 days. The mixture wasthen decanted and the residual solid was dried under vacuum to give 101g (94% yield) of the title intermediate as a crystalline white solid.

¹H NMR (DMSO-d₆, 300 MHz): δ 1.35-1.39 (s, 9H), 1.91-1.95 (m, 2H),2.99-3.04 (t, 2H), 3.43-3.52 (t, 2H), 6.95-6.99 (t, 1H).

Step 2—Preparation of Ethyl(Z)-2-(2-Triphenylmethylaminothiazol-4-yl)-2-(3-N—BOC-aminopropoxyimino)acetate(i.e. ethyl ester of Compound 5a where R¹ is —(CH₂)₃—, —R² is hydrogen,R⁹ is triphenylmethyl, R¹¹ is BOC, and A is hydrogen)

Ethyl(Z)-2-(2-triphenylmethylamino)thiazol-4-yl)-2-(hydroxyimino)acetatehydrochloride (100 g, 202.4 mmol) was dissolved in 700 mL of anhydrousDMF. To this stirred mixture was added cesium carbonate (230.8 g, 708.5mmol) followed by tetrabutylammonium iodide (18.7 g, 50.6 mmol).N—BOC-3-bromopropylamine (50.6 g, 212.5 mmol) in DMF (100 mL) was thenadded dropwise over 30 minutes. The mixture was stirred for two hours atwhich time HPLC indicated that the reaction was complete. The mixturewas then filtered and the filter cake was washed with 200 mL of DMF. Thefiltrate was dissolved in 2 L of ethyl acetate and washed with 700 mL of1N HCl, followed by 700 mL of saturated aqueous sodium bicarbonate, andfinally with 500 mL of brine. The organic layer was dried over sodiumsulfate, filtered, and concentrated under vacuum. The residual oil wasdissolved in 250 mL of boiling ethanol and poured into a beaker. Oncethe material had completely cooled, the residual clay-like solid wasplaced in a Büchner funnel and washed with 50 mL of ethanol previouslycooled to −20° C. (NOTE: the product is moderately soluble in ethanoland use of larger amounts will decrease the overall yield of finalproduct). After air-drying, the residual solid was ground into a finepowder in a mortar and pestle and dried under vacuum to give 117 g (94%yield) of the title intermediate as a fine off-white powder.

¹H NMR (DMSO-d₆, 300 MHz): δ 1.01-1.1 (t, 3H), 1.31 (s, 9H), 1.60-1.70(t, 2H), 2.94-2.99 (m, 2H), 3.95-4.04 (m, 4H), 6.77-6.81 (t, 1H), 6.95(s, 1H), 7.16-7.38 (m, 15H), 8.80 (s, 1H). MS m/z: 615.4 [M+H]⁺.

Step 3—Preparation of(Z)-2-(2-Triphenylmethylaminothiazol-4-yl)-2-(3-N—BOC-aminopropoxyimino)acetate(i.e, Compound 5a where R¹ is —(CH₂)₃—, R² is hydrogen, R⁹ istriphenylmethyl, R¹¹ is BOC, and A is hydrogen)

The ethyl ester from Step 2 above (84.2 g, 137 mmol) was suspended in400 mL of anhydrous ethanol and heated in an oil bath to 80° C. withstirring. After all material had dissolved, potassium hydroxide (23.1 g,411 mmol) in 150 mL of ethanol was added dropwise to the solution over20 minutes. A precipitate began forming 10 minutes after addition of thebase was complete, and within another 10 minutes the mixture was solid.The mixture was removed from the oil bath and cooled in an ice bath.Ethyl acetate and water were added to the cooled mixture which was thenpoured into a separatory funnel. The mixture was washed with 1Nphosphoric acid, which caused the formation of a white solid (NOTE:washing the product with a stronger acid, such as 1N HCl causesdegradation of the product). Water was added to the separatory funnel todissolve this solid, and the organic layer was then washed withsaturated aqueous sodium bicarbonate and with brine. The organic layerwas dried over sodium sulfate, filtered and concentrated under vacuum togive the title intermediate (80 g, 99% yield) as a dark tan solid.

Step 4—Preparation of(Z)-2-(2-Triphenylmethylamino-5-chlorothiazol-4-yl)-2-(3-N—BOC-aminopropoxyimino)acetate(i.e. Compound 5b where R¹ is —(CH₂)₃—, R² is hydrogen, R⁹ istriphenylmethyl, R¹¹ is BOC, and A is chloro)

The intermediate from Step 3 above (10 g, 17.04 mmol) was dissolved in70 mL of chloroform and stirred while solid N-chlorosuccinimide (2.28 g,17.04 mmol) was added (NOTE: experiments suggest that excess NCS mayproduce undesired side products). The mixture was stirred overnight (aminimum of 15 hours) at which time HPLC indicated that the reaction wascomplete. The mixture was then concentrated under vacuum and the residuewas dissolved in a minimal amount of DMF. This mixture was added tovigorously stirred water to form a precipitate which was then collectedby filtration. The solid was air-dried to give 9.5 g (90% yield) of thetitle intermediate as a tan solid. ¹HNMR indicated only a minimal amountof succinimide remaining (NOTE: isolation of chlorinated product is notnecessary for successful coupling in next step, but experiments suggestthat residual succinimide may interfere with subsequent pyridinedisplacement). Alternatively, after the chlorination reaction wascomplete, the reaction mixture was washed with water (3×), brine, andthen dried over anhydrous sodium sulfate. This solution was thenfiltered and concentrated under vacuum to give the title intermediate(90%) as a tan solid.

¹H NMR (DMSO-d₆, 300 MHz): δ 1.37 (s, 9H), 1.63-1.74 (t, 2H), 2.94-2.99(m, 2H), 3.97-4.05 (t, 2H), 6.80-6.85 (t, 1H), 7.18-7.41 (m, 15H), 8.97(s, 1H). MS m/z 621.3 [M+H]⁺.

Step 5—Preparation of(7R)-7-[(Z)-2-(2-Triphenylmethylamino-5-chlorothiazol-4-yl)-2-(3-N—BOC-aminopropoxyimino)acetamido]-3-chloromethyl-3-cephem-4-carboxylatep-Methoxybenzyl Ester (i.e, Compound 7 where R¹ is —(CH₂)₃—, R² ishydrogen, R⁹ is triphenylmethyl, R¹¹ is BOC, and R¹² is p-methoxybenzyl)

The intermediate from Step 4 (0.62 g, 1 mmol) was dissolved in 6 mL ofanhydrous THF, and to this mixture was added 0.34 g (0.83 mmol) of7-amino-3-chloromethylcephalosporanic acid p-methoxybenzyl esterhydrochloride (i.e, compound 6 where R¹² is PMB; obtained from Otsuka,Japan) in 4 mL of anhydrous THF. The resulting mixture was stirred undernitrogen and cooled to −35° C. To this cooled mixture was addeddiisopropylethylamine (0.52 mL, 3 mmol) followed by phosphorousoxychloride (0.11 mL, 1.2 mmol). This mixture was stirred at −20° C. for30 minutes and then quenched with wet THF and diluted with ethylacetate. This mixture was washed with water, 1N HCl, brine, dried oversodium sulfate, filtered and concentrated to give 0.88 g (100% yield) ofthe title intermediate as a brown-red solid. ¹HNMR indicated noundesired isomerization and no residual succinimide.

¹H NMR (DMSO-d₆, 300 MHz): δ 1.37 (s, 9H), 1.63-1.74 (t, 2H), 2.94-2.99(m, 2H), 3.4-3.74 (q, 2H), 3.75 (s, 3H), 3.97-4.05 (t, 2H), 4.40-4.59(q, 2H), 5.11-5.25 (m, 3H), 5.49-5.54 (m, 1H), 6.75-6.81 (t, 1H),6.90-6.96 (d, 2H), 7.18-7.41 (m, 17H), 8.97 (s, 11H), 9.41-9.44 (d, 1H).MS m/z 972.0 [M+H]⁺.

(NOTE: Experiments suggest that DIPEA causes isomerization when theabove reaction is carried out on larger scales. A modified procedurewhich uses 2,4,6-collidine as the base and which maintains thetemperature at −35° C. for the entire course of the reaction—about 10minutes—avoids this problem).

Step 6—Preparation of(7R)-7-[(Z)-2-(2-Triphenylmethylamino-5-chlorothiazol-4-yl)-2-(3-N—BOC-aminopropoxyimino)acetamido]-3-[(1-pyridinio)methyl]-3-cephem-4-carboxylatep-Methoxybenzyl Ester (i.e Compound 8 where R¹ is —(CH₂)₃—, R² ishydrogen, R⁹ is triphenylmethyl, R¹¹ is BOC, R¹² is p-methoxybenzyl andm is 0)

The intermediate from Step 5 (500 mg, 0.514 mmol) was dissolved in 2 mLof anhydrous acetone and protected from light using foil. The solutionwas stirred under a nitrogen atmosphere and 77 mg (0.514 mmol) of sodiumiodide was added and the resulting mixture was stirred for 1 hour.Pyridine (63 μL, 0.772 mmol) was added and, after 90 minutes, themixture was added to 25 mL of ethyl ether. This mixture was centrifugedand the resulting pellet was washed with ethyl ether and centrifugedagain. The ether was decanted and the pellet was dried under vacuum togive a quantitative yield of the title intermediate as a tan solid whichwas used without further purification.

¹H NMR (DMSO-d₆, 300 MHz): δ=1.37 (s, 9H), 1.63-1.74 (t, 2H), 2.94-2.99(m, 2H), 3.3-3.50 (q, 2H), 3.4-3.74 (q, 2H), 3.75 (s, 3H), 3.97-4.05 (t,2H), 5.10-5.12 (d, 1H), 5.21 (s, 2H), 5.50-5.55 (m, 1H), 5.6 (s, 2H),6.75-6.81 (t, 1H), 6.90-6.96 (d, 2H), 7.18-7.41 (m, 17H), 8.16-8.21 (t,2H), 8.61-8.70 (t, 1H), 8.96 (s, 1H), 8.98-9.02 (d, 2H), 9.41-9.44 (d,11H). MS m/z 1014.2 [M+H]⁺.

Step 7—Preparation of(7R)-7-[(Z)-2-(2-Amino-5-chlorothiazol-4-yl)-2-(3-aminopropoxyimino)acetamido]-3-[(1-pyridinio)methyl]-3-cephem-4-carboxylateBis-Trifluoroacetic Acid Salt (i.e, Compound 2 where R¹ is —(CH₂)₃—, R²is hydrogen and m is 0)

The intermediate from Step 6 (14.4 g) was dissolved in a 1:1 mixture oftrifluoroacetic acid and dichloromethane (120 mL). To this stirringmixture was added 6.2 mL of anisole and the resulting mixture wasstirred for 3 hours at room temperature. The mixture was thenconcentrated and the residue dissolved in ethyl acetate and extractedwith water. The water layers were lyophilized and the resulting powderwas dissolved in water and purified using reverse-phase prep HPLC. Theresulting purified aqueous solution was then lyophilized to give 3.3 g(30% yield) of the title intermediate.

¹H NMR (DMSO-d₆, 300 MHz): δ=1.80-1.97 (t, 2H), 2.79-2.92 (m, 2H),3.29-3.57 (q, 2H), 4.02-4.15 (t, 2H), 5.15-5.19 (d, 1H), 5.41-5.63 (q,2H), 5.83-5.92 (m, 1H), 7.39 (s, 2H), 7.77 (s, 3H), 8.17-8.22 (t, 2H),8.60-8.70 (t, 1H), 9.0-9.08 (d, 2H), 9.59-9.62 (d, 1H). MS m/z 553.1[M+H]⁺.

(NOTE: The above reaction can also be conducted using triethylsilane inplace of the anisole. Additionally, the product can be isolated usingethyl ether trituration).

Example B Synthesis of(7R)-7-[(Z)-2-(2-Amino-5-chlorothiazol-4-yl)-2-(3-aminopropoxyimino)acetamido]-3-[(2,3-cyclopenteno-1-pyridinio)methyl]-3-cephem-4-carboxylatebis-Trifluoroacetic Acid Salt

Using the procedure described in Example A and substituting2,3-cyclopentenopyridine (obtained from Koei, Japan) for pyridine inStep 6, the title intermediate was obtained.

¹H NMR (DMSO-d₆, 300 MHz): δ=1.82-1.947 (t, 2H), 2.18-2.29 (m, 2H),2.40-2.58 (m, 2H), 2.81-2.95 (m, 2H), 3.09-3.17 (t, 2H), 3.21-3.30 (t,2H), 4.10-4.19 (t, 2H), 5.15-5.19 (d, 1H), 5.40-5.61 (q, 2H), 5.83-5.92(m, 1H), 7.39 (s, 2H), 7.77 (s, 3H), 7.89-7.96 (t, 2H), 8.42-8.48 (d,1H), 8.62-8.69 (d, 1H), 9.60-9.63 (d, 1H). MS m/z 592.5 [M+H]⁺.

Example C Synthesis of(7R)-7-[(Z)-2-(2-Amino-5-chlorothiazol-4-yl)-2-(6-aminohexoxyimino)acetamido]-3-[(1-pyridinio)methyl]-3-cephem-4-carboxylatebis-Trifluoroacetic Acid Salt

Using the procedure described in Example A and substitutingN—BOC-6-iodohexylamine for N—BOC-3-bromopropylamine in Step 2 (andeliminating the tetrabutylammonium iodide), the title intermediate wasobtained.

¹H NMR (DMSO-d₆, 300 MHz): δ 1.2 ppm (bs, 4H), 1.3 ppm (m, 2H), 1.5 ppm(m, 2H), 2.7 ppm (m, 2H), 3.3 ppm (dd, 2H), 4.0 ppm (t, 3H), 5.1 ppm (d,1H), 5.5 ppm (dd, 2H), 5.8 ppm (dd, 1H), 7.25 ppm (bs, 2H), 7.6 ppm (bs,3H), 8.2 ppm (dd, 2H), 8.6 ppm (dd, 1H), 9 ppm (dd, 2H), 9.5 ppm (d,1H). MS m/z 594.3 (M+).

Example D Synthesis of(7R)-7-[(Z)-2-(2-Amino-5-chlorothiazol-4-yl)-2-(2-(2-aminoethoxy)ethoxyimino)acetamido]-3-[(1-pyridinio)methyl]-3-cephem-4-carboxylatebis-Trifluoroacetic Acid Salt

The procedure of Example A was used, except that the following procedurewas substituted for Step 2:

Step 2—Preparation of Ethyl(Z)-2-(2-Triphenylmethylaminothiazol-4-yl)-2-[2-(2-N—BOC-aminoethyl)ethoxyimino]acetate(i.e, ethyl ester of Compound 5a where R¹ is —(CH₂)₂—O—(CH₂)₂—, R² ishydrogen, R⁹ is triphenylmethyl, R¹¹ is BOC, and A is hydrogen)

The intermediate from Step 1 in Example A (42.5 g, 86 mmol) was added toa stirred suspension of N—BOC-2-(2-iodoethoxy)-ethylamine (28.5 g, 90mmol) (prepared in three steps from 2-(2-hydroxyethoxy)ethanol, i.e.,(i) BOC₂O, KOH, (ii) MsCl, Et₃N and (iii) NaI) and cesium carbonate(84.1 g, 258 mmol) in DMF (300 mL). The suspension was stirred for 16 hat room temperature at which time HPLC indicated that the reaction wascomplete. The reaction mixture was then filtered and the filter cakewashed with DMF (100 mL). The filtrate was diluted with ethyl acetate (1L) and washed with water (300 mL), 1N HCl (200 mL), saturated aqueoussodium bicarbonate (200 mL) and brine (200 mL). The organic layer wasdried over magnesium sulfate, filtered, and concentrated in vacuo. Theresidue was purified by flash column chromatography (ethylacetate:hexane, 1:1) to afford 49.7 g (90% yield) of the titleintermediate as an off-white solid.

¹H NMR (DMSO-d₆, 300 MHz): δ=2.96 (s, broad, 2H), 3.20-3.55 (q, 2H),3.59 (t, 2H), 3.70 (t, 2H), 4.19 (t, 2H), 5.13 (d, 1H), 5.31-5.64 (q,2H), 5.80 (dd, 1H), 7.40 (s, 2H), 7.87 (s, broad, 3H), 8.20 (t, 2H),8.64 (t, 1H), 9.23 (d, 2H), 9.55 (d, 1H). MS m/z 503.1 [M-pyridine]⁺.

Example E Synthesis of(7R)-7-[(Z)-2-(2-Amino-5-chlorothiazol-4-yl)-2-(4-aminomethylbenzyloxyimino)acetamido]-3-[(1-pyridinio)methyl]-3-cephem-4-carboxylatebis-Trifluoroacetic Acid Salt

Using the procedure described in Example A and Step 2 of Example D andsubstituting N—BOC-4-(iodomethyl)benzylamine (prepared in four step from4-(aminomethyl)benzoic acid, i.e., (i) BOC₂O, KOH, (ii) LiAlH₄, (iii)MsCl, Et₃N and (iv) NaI) for N—BOC-2-(2-iodoethoxy)-ethylamine3-bromopropylamine hydrobromide in Step 2, the title intermediate wasobtained.

¹H NMR (DMSO-d₆, 300 MHz): δ=3.18-3.59 (q, 2H), 4.00 (s, broad, 2H),5.13 (s, 2H), 5.15 (d, 2H), 5.40-5.64 (q, 2H), 5.85 (dd, 1H), 7.38-7.43(m, 6H), 8.19-8.23 (m, 4H), 8.64 (t, 1H), 9.17 (d, 2H), 9.71 (d, 11H).MS m/z 614.1 [M+H]⁺, 535.1 [M-pyridine]⁺.

Example F (Comparative) Synthesis of(7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(3-aminopropoxyimino)acetamido]-3-[(1-pyridinio)methyl]-3-cephem-4-carboxylatebis-Trifluoroacetic Acid Salt

By eliminating Step 4 in Example A above, the des-chloro derivative ofthe intermediate of Example A was prepared.

¹H NMR (DMSO-d₆, 300 MHz): δ=1.75-1.82 (t, 2H), 2.67-2.82 (m, 2H),3.25-3.61 (q, 2H), 3.98-4.09 (t, 2H), 5.13-5.17 (d, 1H), 5.38-5.58 (q,2H), 5.79-5.85 (m, 1H), 6.62 (s, 1H) 7.15-7.25 (s, broad, 2H), 7.60-7.75(s, broad, 3H), 8.16-8.19 (t, 2H), 8.58-8.63 (t, 1H), 8.95-9.01 (d, 2H),9.57-9.60 (d, 1H). MS m/z 518.6 [M+H]⁺.

Example G Synthesis of Vancomycin 3-(Aminooxy)propyl Amide

Step 1—Preparation of N-(3-Aminopropoxy)phthalimide

N-(tert-Butoxycarbonyl)-3-bromopropylamine (from Step 1 in Example Aabove) (9.58 g, 40.23 mmol) and N-hydroxyphthalimide (6.36 g, 39 mmol)were dissolved in 70 mL of anhydrous DMF. To this solution was addeddiisopropylethylamine (7.01 mL, 40.23 mmol) resulting in a deep redcolor. The reaction was stirred at room temperature for 16 hours afterwhich time, the reaction mixture was poured into 500 mL of diethylether. The resulting white precipitate was filtered off and discarded.The organic solution was washed with 2×200 mL saturated sodiumbicarbonate and with 2×200 mL of water. The organic solution was driedover anhydrous magnesium sulfate, filtered and concentrated to give awhite solid. This solid was then dissolved in 50 mL of DCM and 50 mL ofTFA. After stirring for 1 hour, this solution was poured into 300 mL ofdiethyl ether. The resulting precipitate was filtered, washed withdiethyl ether and dried under vacuum to afford the title intermediate asits trifluoroacetic acid salt.

¹H NMR (DMSO-d₆, 300 MHz): δ=1.90 (2H, qn), 2.95 (2H, t), 4.18 (2H, t),7.79 (4H, s), 7.92 (3H, broad s).

Step 2—Preparation of Vancomycin 3-(Phthalimidooxy)propyl Amide

Vancomycin hydrochloride (10.0 g, 6.74 mmol) and the intermediate fromStep 1 (2.70 g, 8.09 mmol) were slurried in 100 mL of anhydrous DMF.Diisopropylethylamine (4.70 mL, 26.98 mmol) was added and the resultingmixture was stirred at room temperature for 10 min. A solution of PyBOP(5.61 g, 10.78 mmol) and HOAt (1.65 g, 10.78 mmol) in DMF (20 mL) wasthen added, and the reaction was stirred at room temperature. After 1hour, the reaction mixture was added to diethyl ether (500 mL). Theresulting precipitate was filtered, washed with diethyl ether and driedunder vacuum to yield the title intermediate as an off-white solid.

MS m/z=1651.8 (M+H)⁺.

Step 3—Preparation of Vancomycin 3-(Aminooxy)propyl Amide

The intermediate from Step 2 (11.2 g, 6.74 mmol) was slurried in 80 mLof anhydrous DMF and hydrazine monohydrate (0.65 mL, 13.48 mmol) wasadded. The reaction was stirred at room temperature for 4.5 hours andthen 1 mL of trifluoroacetic acid was added to the reaction mixture,followed by 300 mL of diethyl ether. After vigorous stirring, theresulting precipitate was filtered, washed with diethyl ether and driedunder vacuum. The title compound was purified by reverse phase HPLCusing a water/methanol gradient to afford the title intermediate as alyophilized powder.

MS m/z=1522.9 (M+H)⁺.

Example H Synthesis of(7R)-7-[2-(2-Amino-5-chlorothiazol-4-yl)-2-oxoacetamido]-3-(1-pyridinio)methyl-3-cephem-4-carboxylateBis-Trifluoroacetate

Step 1—Preparation of Ethyl2-(2-Formylamino-5-chlorothiazol-4-yl-2-oxoacetate

Ethyl 2-(Formylaminothiazol-4-yl)-2-oxoacetate (9.1 g, 39.87 mmol)(obtained from Aldrich, Milwaukee, Wis.) was slurried in 50 mL ofanhydrous DMF. N-Chlorosuccinimide (5.6 g, 41.86 mmol) was added as asolid and the suspension was stirred at room temperature. After 18hours, the reaction mixture was poured into 500 mL of water. Theresulting white precipitate was filtered, washed with water and airdried to afford the title intermediate as a white solid.

¹H NMR (DMSO-d₆, 300 MHz): δ=1.2 (t, 3H), 4.3 (q, 2H), 8.55 (s, 1H).

Step 2—Preparation of 2-(2-Formylamino-5-chlorothiazol-4-yl)-2-oxoaceticAcid

To the intermediate from Step 1 (3.6 g, 13.7 mmol) was added 1M NaOH (30mL, 30 mmol). The resulting suspension was stirred at room temperaturefor 2 hours (at which time the solution was clear) and 1M HCl (30 mL, 30mmol) was then added, followed by 100 mL of water. After vigorousstirring, the resulting precipitate was filtered, washed with a minimumamount of cold water and air dried to afford the title intermediate asan off-white solid.

¹H NMR (DMSO-d₆, 300 MHz): δ=8.5 (s, 1H).

Step 3—Preparation of(7R)-7-[2-(2-Formylamino-5-chlorothiazol-4-yl)-2-oxoacetamido]-3-chloromethyl-3-cephem-4-carboxylatep-Methoxybenzyl Ester

The intermediate from Step 2 (1.03 g, 4.37 mmol),7-amino-3-chloromethylcephalosporanic acid p-methoxybenzyl esterhydrochloride (1.95 g, 4.81 mmol) and HOAt (0.74 g, 4.81 mmol) wereslurried in 15 mL of anhydrous DMF. The reaction vessel was purged withnitrogen and then cooled to 0° C. with an external ice bath.1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.92 g,4.81 mmol) was added to the cold reaction mixture, followed by2,4,6-collidine (0.64 mL, 4.81 mmol). The reaction was stirred at 0° C.for 2 hours and then poured into 200 mL of 0.5M HCl. The resultingprecipitate was filtered, washed with water and air dried to afford thetitle intermediate as a red solid. The compound was used without furtherpurification.

MS m/z=607 (M+Na)⁺.

Step 4—Preparation of(7R)-7-[2-(2-Formylamino-5-chlorothiazol-4-yl)-2-oxoacetamido]-3-(1-pyridinio)methyl-3-cephem-4-carboxylatep-Methoxybenzyl Ester

The intermediate from Step 3 (2.5 g, 4.27 mmol) and sodium iodide (0.64g, 4.27 mmol) were dissolved in acetone and shielded from light by foil.The reaction was stirred for 10 minutes and then pyridine (0.42 mL, 5.12mmol) was added. The reaction was then stirred at room temperature for 1hour and then 300 mL of water were added. The resulting precipitate wasfiltered, washed with water and air-dried to provide a red solid. Thissolid was purified on reverse-phase HPLC and the resulting aqueoussolution was lyophilized to afford the title intermediate as alyophilized powder.

MS m/z=628.1 (M)⁺.

Step 5—Preparation of(7R)-7-[2-(2-Amino-5-chlorothiazol-4-yl)-2-oxoacetamido]-3-(1-pyridinio)methyl-3-cephem-4-carboxylateBis-Trifluoroacetate

The intermediate from Step 4 (0.11 g, 0.18 mmol) was dissolved in 5 mLof methanol and concentrated aqueous hydrochloric acid (0.5 mL) wasadded. The resulting solution was stirred at room temperature for 1.5hours. The methanol was removed under vacuum and acetonitrile (10 mL)was added. The solution was then concentrated in vacuum and to theresidue as added DCM (2 mL) and TFA (2 mL) and the resulting mixture wasstirred at room temperature for 1.5 hours. Diethyl ether (50 mL) wasthen added and the title intermediate was isolated by centrifugation.This intermediate was used without further purification.

MS m/z=479.9 (M)⁺.

Example I Synthesis of Compound 13 where R¹ is —(CH₂)₃—, R², R⁵, R⁶, R⁸are hydrogen, R⁴ is hydroxy, R⁷ is methyl, X¹ and X² are chloro, and mis 0

Step 1—Preparation of(Z-2-(2-Amino-5-chlorothiazol-4-yl)-2-(3-aminopropoxyimino)acetate

The intermediate from Step 4 in Example A (0.75 g, 1.21 mmol) wasdissolved in 5 mL of DCM and 5 mL of trifluoroacetic acid. After 1 hourstirring at room temperature, 100 mL of diethyl ether was added. Theresulting precipitate was filtered, washed with diethyl ether and driedunder vacuum to afford the title intermediate as a brown solid

Step 2—Preparation of Compound 13 where R¹ is —(CH₂)₃—, R², R⁵, R⁶, R⁸are hydrogen R⁴ is hydroxy R⁷ is methyl, X¹ and X² are chloro, and m is0

Vancomycin hydrochloride (1.3 g, 0.88 mmol) and HOAt (0.14 g, 0.088mmol) were slurried in 3.5 mL of anhydrous DMSO. A solution of PyBOP(0.46 g, 0.88 mmol) in 3.5 mL of anhydrous DMF was added, followed byDIPEA (154 uL, 0.88 mmol). After stirring for 20 minutes, a solution ofthe intermediate from Step 1 (0.22 g, 0.44 mmol) in 1 mL of DMF wasadded, followed quickly by addition of DIEA (0.54 mL, 3.08 mmol). Thereaction mixture was stirred at room temperature for 1 hour, then 0.5 mLof trifluoroacetic acid was added, followed quickly by the addition of100 mL of Et₂O. The resulting precipitate was filtered, washed with Et₂Oand dried in vacuum. The crude product was purified byreverse-phase-HPLC and the resulting aqueous solution was lyophilized toafford the title intermediate as a lyophilized powder.

MS m/z=1711.0 (M+H)⁺.

Example 1 Synthesis of a Compound of Formula I where R¹ is —(CH₂)₃—, R²,R⁵, R⁶, R⁸ are hydrogen, R⁴ is hydroxy, R⁷ is methyl, X¹ and X² arechloro, and m is 0 (Compound 1 in Table I)

Vancomycin hydrochloride (4.2 g, 2.8 mmol) was dissolved in 40 mL ofDMSO. To this solution was added a solution of PyBOP (1.3 g, 2.6 mmol)and HOAT (0.35 g, 2.6 mmol) in 40 mL of DMF, followed by 0.98 mL (5.68mmol) of diisopropylethylamine. This mixture was stirred at roomtemperature for 30 minutes and then quenched with 0.44 mL (5.7 mmol) oftrifluoroacetic acid. The mixture was then cooled to 0° C. and asolution of the intermediate from Example A above (1.3 g, 2.6 mmol) in20 mL of DMF at 0° C. was added, followed by 1.5 mL (11.4 mmol) of2,4,6-collidine. The mixture was maintained at 0° C. for four hours andthen quenched with 1.1 mL of trifluoroacetic acid. This mixture was thenadded to ethyl ether to form a precipitate, centrifuged, washed withether, decanted and dried under vacuum. The resulting powder wasdissolved in water and purified using prep HPLC. The fractionscontaining the desired product were lyophilized to give thetri-trifluoroacetic acid salt of the title compound. The anion of thesalt was then exchanged using Amberlyte resin to afford thetri-hydrochloride salt of the title compound (1.4 g, 27% yield) as awhite powder.

MS m/z 953.3 [[M+H]⁺-pyridine]/2; 992.0 [M+H]⁺/2.

Additionally, Compounds 2-30 shown in Table 1 are or were prepared usingthe procedures of Example A and Example I by using in place of thepyridine in Step 6 of Example A, the following substituted pyridines:

Example 2-2-Picoline

Example 3-3-Picoline

Example 4-4-Picoline

Example 5-2-Methoxypyridine

Example 6-3-Methoxypyridine

Example 7-4-Methoxypyridine

Example 8-2-Thiomethoxypyridine

Example 9-3-Thiomethoxypyridine

Example 10-4-Thiomethoxypyridine

Example 11-2-Fluoropyridine

Example 12-3-Fluoropyridine

Example 13-4-Fluoropyridine

Example 14-2-Chloropyridine

Example 15-3-Chloropyridine

Example 16-4-Chloropyridine

Example 17-2-Phenylpyridine

Example 18-3-Phenylpyridine

Example 19-4-Phenylpyridine

Example 20-4-Cyclopropylpyridine

Example 21-4-(Carboxythiomethoxy)pyridine

Example 22-Isonicotinamide

Example 23-2,3-Lutidine

Example 24-3,4-Lutidine

Example 25-3,5-Lutidine

Example 26-3,4-Dimethoxypyridine

Example 27-4-Methoxy-3-methylpyridine

Example 28-4-Fluoro-3-methoxypyridine

Example 29-2,3-Cyclohexenopyridine

Example 30-2,3-Cyclopentenopyridine

The above substituted pyridines are either commercially available or canbe prepared by literature procedures.

Example 31 Synthesis of a Compound of Formula I where R¹ is —(CH₂)₆—,R², R⁵, R⁶, R⁸ are hydrogen, R⁴ is hydroxy, R⁷ is methyl, X¹ and X² arechloro, and m is 0 (Compound 31 in Table I)

Using the procedure of Example 1 and substituting the intermediate ofExample C for the intermediate of Example A, the title compound wasprepared.

MS m/z 2026.5 (M+).

Example 32 Synthesis of a Compound of Formula I where R¹ is—(CH₂)₂—O—(CH₂)₂—, R², R⁵, R⁶, R⁸ are hydrogen, R⁴ is hydroxy, R⁷ ismethyl, X¹ and X² are chloro, and m is 0 (Compound 32 in Table I)

Using the procedure of Example 1 and substituting the intermediate ofExample D for the intermediate of Example A, the title compound wasprepared.

MS m/z 967.9 [(M-pyridine)/2]⁺.

Example 33 Synthesis of a Compound of Formula I where R¹ is —CH₂-1,4-Ph—CH₂—, R², R⁵, R⁶, R⁸ are hydrogen, R⁴ is hydroxy, R⁷ is methyl, X¹ andX² are chloro, and m is 0 (Compound 33 in Table I)

Using the procedure of Example 1 and substituting the intermediate ofExample E for the intermediate of Example A, the title compound wasprepared.

MS m/z 1967.0 [M+H]⁺, 984.2 [(M-pyridine)/2]⁺.

Example 34 (Comparative) Synthesis of a des-Chloro Compound of Formula Iwhere R¹ is —(CH₂)₃—, R², R⁵, R⁶, R⁸ are hydrogen, R⁴ is hydroxy, R⁷ ismethyl, X¹ and X² are chloro, and m is 0 (Compound 34)

Using the procedure of Example I and substituting the des-chlorocephalosporin intermediate of Example F for the intermediate of ExampleA, the title compound was prepared.

MS m/z 935.3 [[M+H]⁺-pyridine]/2; 974.9 [M+H]⁺/2.

Example 35 Determination of Minimal Inhibitory Concentrations (MICs)

Minimal inhibitory concentration (MICs) assays were performed using thebroth microdilution method set forth in NCCLS guidelines (see, NCCLS.2000. Methods for Dilution Antimicrobial Susceptibility Tests forBacteria That Grow Aerobically; Approved Standard—Fifth Ed., Vol. 20,No. 2). Bacterial strains were obtained from the American Type TissueCulture Collection (ATCC), Stanford University Hospital (SU), KaiserPermanente Regional Laboratory in Berkeley (KPB), Massachusetts GeneralHospital (MGH), the Centers for Disease Control (CDC), the San FranciscoVeterans' Administration Hospital (SFVA) or the University of CaliforniaSan Francisco Hospital (UCSF). Vancomycin-resistant enterococci werephenotyped as Van A or Van B based on their sensitivity to teicoplanin.Some vancomycin-resistant enterococci that had been genotyped as Van A,Van B, Van C1 or Van C2 were also obtained from the Mayo Clinic.

In this assay, cryopreserved bacterial cultures of reference andclinical strains were streaked for isolation on appropriate agar medium(i.e., Trypticase Soy Agar, Trypticase Soy Agar with defibrinated sheeperthrocytes, Brain Heart Infusion Agar, Chocolate Agar). Followingincubation to allow formation of colonies, these plates were sealed withparafilm and stored refrigerated for up to two weeks. For preparation ofassay inocula and to ensure low variability, several colonies from abacterial isolate cultured on the agar plates were pricked with aninoculating loop and aseptically transferred to Mueller-Hinton Broth(supplemented with divalent cations to required levels based onmanufacturer's certification). The broth culture was grown overnight at35° C., diluted in fresh prewarmed broth and grown to log phase; this isequivalent to a 0.5 MacFarland standard or 1×10⁸ colony forming unitsper milliliter (CFU/mL). Not all cell suspensions, due to speciesvariability, contained 1×10⁸ CFU/mL when turbidity is equivalent to theMacFarland standard, therefore acceptable adjustments (based on NCCLSguidelines) were made in dilutions of different bacterial strains. Theinoculum was diluted such that 100 μL of this culture in Mueller-HintonBroth, supplemented Mueller-Hinton Broth, or Haemophilus test medium,when over layered onto a 2-fold serially diluted series of antibioticconcentrations also in 100 μL of corresponding medium, in a 96-wellmicrotiter plate resulted in a starting bacterial concentration of 5×10⁵CFU/mL. The plates were then incubated 18-24 hours at 35° C. The MIC wasread visually as the lowest concentration well with no bacterial growth.Bacterial growth is defined as more than three pinpoint colonies, abutton of precipitated cells larger than 2 mm in diameter, or obviousturbidity.

Strains routinely tested in the initial screen includedmethicillin-sensitive Staphylococcus aureus (MSSA),methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcusaureus producing penicillinase, methicillin-sensitive Staphylococcusepidermidis (MSSE), methicillin-resistant Staphylococcus epidermidis(MRSE), vancomycin-sensitive Enterococcus faecium (EFMVS),vancomycin-sensitive Enterococcus faecalis (EFSVS), vancomycin-resistantEnterococcus faecium also resistant to teicoplanin (EFMVR Van A),vancomycin-resistant Enterococcus faecium sensitive to teicoplanin(EFMVR Van B), vancomycin-resistant Enterococcus faecalis also resistantto teicoplanin (EFSVR Van A), vancomycin-resistant Enterococcus faecalissensitive to teicoplanin (EFSVR Van B), penicillin-sensitiveStreptococcus pneumoniae (PSSP) and penicillin-resistant Streptococcuspneumoniae (PSRP). Because of the inability of PSSP and PSRP to growwell in Mueller-Hinton broth, MICs with those strains were determinedusing either TS broth supplemented with defibrinated blood orHaemophilus test medium.

Test compounds having significant activity against the strains mentionedabove were then tested for MIC values in a larger panel of clinicalisolates including the species listed above as well as non-speciatedcoagulase negative Staphylococcus both sensitive and resistant tomethicillin (MS-CNS and MR-CNS). Additionally, these test compounds werealso assayed for MICs against gram-negative microorganisms, such asEscherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae,Enterobacter cloacae, Acinetobacter baumannii, Haemophilus influenzaeand Moraxella catarrhalis.

Table II shows MIC₉₀ data for a compound of this invention againstmethicillin-resistant S. aureus (MRSA) and methicillin-resistant S.epidermitis (MRSE) as compared to the known antibiotic, vancomycin.

TABLE II Minimum Inhibitory Concentrations (MICs) Test MIC₉₀ ²Microorganism Compound (μg/mL) Methicillin-resistant S. aureus (MRSA)Compound 1 <0.1 (n = 53)¹ Vancomycin 3 Methicillin-resistant S.epidermitis (MRSE) and Compound 1 <0.1 other coagulase-negativestaphylococci (n = 34) Vancomycin 4 ¹Number of strains tested. ²Minimuminhibitory concentration for 90% of strains tested.

Additionally, as shown in Table III, compounds of this invention alsohad surprising and unexpected MICs against various methicillin-resistantS. aureus strains when compared to a related des-chloro derivative(i.e., Compound 34).

TABLE III Minimum Inhibitory Concentrations MIC (μg/mL) MicroorganismCmpd. 1 Cmpd. 34 MRSA 33591 ≦0.1 0.17 MRSA MED-103 ≦0.1 0.20 MRSAMED-104 0.10 0.58 MRSA MED-107 ≦0.1 0.34 MRSA MED-110 0.20 0.49 MRSAMED-572 ≦0.1 <0.1 MRSA MED-84 ≦0.1 0.29 MRSA MED-85 ≦0.1 0.32 MRSAMED-86 ≦0.1 0.29 MRSA MED-87 ≦0.1 0.49 MRSA MED-88 ≦0.1 0.34 MRSA MED-89≦0.1 0.18

Example 36 Time-Kill Assay

This time-kill assay is a method for measuring the rate of bactericidalactivity of a test compound. These procedures are similar to thosedescribed in V. Lorian, “Antibiotics in Laboratory Medicine”, FourthEdition, Williams and Wilkins (1996), pages 104-105. A rapid time-killis desirable to quickly prevent bacterial colonization and reduce hosttissue damage.

Bacterial inocula were prepared as described in Example 35 fordetermination of MIC. Bacteria were diluted in prewarmed media in shakeflasks and incubated with shaking (200 rpm, 35° C.). At 0, 1, 4, and 24hours samples were withdrawn from the flasks and bacteria wereenumerated by plate counting. Subsequent to the initial sampling, acompound to be assayed was added to the shake flask culture. Platecounts at these intervals previous to and following addition of thecompound were expressed graphically in a time-kill curve. Bactericidalactivity is defined as a ≧3 log decrease (reduction greater than orequal to 99.9%) in bacterial cell numbers by 24 hours.

In this assay, a compound of formula I, i.e., Compound 1, wasbactericidal against MSSA 13709 and MRSA 33591 at a concentration of ≦1μg/mL in 4 hours. By comparison, vancomycin was bactericidal againstMSSA 13709 and MRSA 33591 at a concentration of 4 μg/mL in 24 hours.

Example 37 In vivo Efficacy Studies in Neutropenic Mice

Animals (male CD-1 mice, 20-30 g) were acquired from Charles RiversLaboratories (Gilroy, Calif.) and allowed access to food and water adlibitum. Neutropenia was induced via 200 mg/kg intraperitoneal (IP)injection of cyclophosphamide given four and two days prior to theinoculation of bacteria.

The organism used was either a susceptible or resistant strain ofclinically relevant gram positive pathogens, such asmethicillin-susceptible Staphylococcus aureus (MSSA 13709) andmethicillin-resistant Staphylococcus aureus (MRSA 33591). The bacterialinoculum concentration was ˜10⁶ CFU/mL. Animals were lightlyanesthetized with isoflurane and 50 mL of the bacterial inoculum wasinjected into the anterior thigh. One hour after the inoculation,animals were dosed intravenously with vehicle or the appropriate dose ofthe test compound. At 0 hours and 24 hours post-treatment, the animalswere euthanized (CO₂ asphyxiation) and the anterior and posterior thighcollected aseptically. The thigh was placed into 10 mL sterile salineand homogenized. Dilutions of the homogenate were plated onto tripticsoy agar plates which were incubated overnight. The number of bacterialcolonies on a given plate was multiplied by the dilution factor, dividedby the thigh weight (in grams) and expressed as log CFU/g. ED₅₀ (doserequired to produce 50% of the maximum reduction in thigh titre) wasestimated for each test compound.

In this assay using MRSA 33591, a compound of formula I, i.e., Compound1, had an ED₅₀ of <0.20 mg/kg, iv, compared to an ED₅₀ of 9 mg/kg, iv,for vancomycin.

Example 38 Determination of Aqueous Solubility

The aqueous solubility of a compound of this invention was determinedusing the following procedure. A 5 wt. % dextrose buffer solution at pH2.2 was prepared by adding 1 mL of 1 N hydrochloric acid (Aldrich) to 99mL of a 5 wt. % aqueous dextrose solution (Baxter).

A 1 mg/mL stock solution for calibration standards was then prepared bydissolving 1 mg of the test compound in 1 mL of DMSO. This solution wasvortexed for 30 seconds and then sonicated for 10 minutes. The stocksolution was then diluted with water to prepare calibration standardshaving the following concentrations: 50, 125, 250, 375 and 500 ug/mL.

Each test compound (30 mg) was weighed into a Millipore non-sterile,Ultrafree-MC 0.1 um filter unit (Millipore UFC30VVOO) and a magneticstir bar was added to each unit. The 5 wt. % dextrose buffer solution(750 uL) was then added to each unit and these mixtures were vortexedfor 5 minutes. The filter units were then placed in an Eppendorf tuberack and the tube rack was placed on top of a magnetic stirrer. Eachunit was then titrated to pH 3 using 1 N NaOH (VWR) and the resultingsolutions centrifuged at 7000 rpms for 5 minutes. Each unit was thendiluted 200 fold with 5% dextrose buffer solution and the dilutedsamples were transferred into auto sampler vials for analysis.

The calibration standards and the test samples were analyzed byreverse-phase HPLC using the following conditions:

Column: Luna 150 × 4.6 mm; C18; 5 u Mobile phase: A = 5/95, B = 95/5,both = MeCN/H₂O; 0.1% TFA Method: 10m Lido 100 (0-100% B in 6 min)Injection volume: 20 uL Wavelength: 214 nm

The solubility of each test sample was calculated by comparing the peakarea of the test sample to the calibration curve and multiplying by thedilution factor. Using the above procedure with duplicate samplepreparations, Compound 1 was found to have a solubility of >47.9 mg/mL.

While the present invention has been described with reference tospecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made an equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited herein are incorporated by reference herein intheir entirety to the same extent as if they had been individuallyincorporated by reference.

1. A compound of the formula:


2. A pharmaceutically acceptable salt of a compound of the formula:


3. A hydrochloride salt of a compound of the formula:


4. A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of theformula:


5. A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a pharmaceuticallyacceptable salt of a compound of the formula:


6. A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a hydrochloride saltof a compound of the formula: